This invention relates generally to a collagen material having a form of thin fibrils and a method for obtaining fibril collagen and, more particularly, to a process that reduces native type II collagen fibers into their constituent thin fibrils.
The collagens are fibrous proteins found in all multicellular animals. Collagens are the major component of skin, bones, cartilages, teeth, tendons and other extracellular matrixes (ECM). Collagen is the most abundant protein in mammals (about 25% to 30% of total protein mass). The major collagen types are types I, II, III, V, and XI (collectively 80-90% of all collagen in the body). Collagen molecules are about 300 nm long with rather short nonhelical regions at both ends (N- and C-telopeptides). The collagen molecules form fibers of different size and diameter. Negative and positive staining shows that collagen types I, II, and III each have fiber periodic bands of about 67 nm (Mosher, D. F. et al., “Assembly of extracellular matrix,” Curr. Opin. Cell Biol. 4.5 (1992): 810-818), which correspond to their complicated macromolecular structure.
Collagen is a crucial structural component of almost all connective tissues, and its three-dimensional molecular packing structure and microfibrillar and fibrillar structures are crucial aspects of the proper function of animal tissues. Collagen type II is a major component of articular cartilage, bones, notochord, vitreous humour and other cartilage-like tissues. The type II collagen molecule is a homotrimer with three identical al chains, coded by a single gene; each of them is 1060 amino acid residue long after the cleavage of their pro-peptides. Prockop, D. J. et al., “Collagens: molecular biology, diseases, and potentials for therapy,” Annu. Rev. Biochem. 64 (1995): 403-434. It has a triple helical region about 300 nm long and short nonhelical N- and Ctelopeptides, the triple helix diameter is approximately 1.4 nm and collagen molecular weight is about 285 kDa. The sequence of collagen type II has been identified for some species such as humans (ExPASy sequence data bank code P02458). Sea lamprey (Petromyzon marinus) notochord is a particularly useful source of collagen for structural characterization of collagen molecules and fibrils, due to the fact that collagen packing in the lamprey is relatively crystalline in its notochord relative to that of other animal tissues. The fact that lamprey collagen type II fibrils appear to be indistinguishable from mammalian cartilage as judged by its amino acid staining pattern viewed via TEM (Sheren et al., “Type II collagen of lamprey”, Comp. Biochem Physiol B 85 (1986): 5-14), whilst being organized in a more simplistic manner indicates the tissues usefulness as a model system for mammals. Although the amino acid sequence of collagen type II of sea lamprey is not fully established (only the C-terminal half), it is apparent from TEM data that any differences in the N-terminal half are likely to be trivial (see above). In addition, unlike collagen type I, collagen type II is highly homologous between species. Kadler, K. E., “Extracellular matrix. Fibril-forming collagens,” Protein Profile 2 (1995): 491-619.
Collagen type II molecules are known to form fibrils (which may be made from microfibrils) which bundle to form fibers and can be visualized by microscopy techniques. The type II fibers are different from those of type I collagen, and the higher structures they form can vary between the tissues and species, depending on the particular ECM content. The collagen type II microfibril is believed to be a super coil of five collagen molecules, which is the same as type I collagen, but their fibillar structures are significantly different. Collagen type II fibers are found in cartilage, bones, intervertebral discs, inner ear, vitreous humour, dermis, and notochord. The fibers in these tissues are formed by collagen type II with the participation of other ECM molecules (collagen-ligands) produced by tissue-specific cells. The interaction of the collagen-ligands with collagen influences the size of the fibrils/fibril bundles and their aggregate fibers and meshwork in the tissues. Among these tissue types, lamprey notochord and bovine articular cartilage, for example, contain more collagen type II than any other collagen type, and their aggregates are rather large.
Notochord, a characteristic tissue of chordates, is a cartilage-like tissue that spans the length of the chordate back, located beneath and parallel to the central nervous system between the brain and tail. This tissue is composed of cells embedded in a fibrous sheath, which is in turn composed primarily of collagen, and has a roughly cylindrical shape. Although it is the main axial skeleton at the embryonic stage, the notochord is replaced by the vertebral column in most vertebrates. However, in some chordates it remains into adulthood (e.g., lamprey, lungfish, sturgeon, and some sharks). The mature notochord contains a soft cellular inner part, surrounded by protective fibrous sheath, composed of three layers: inner basal lamina, thick collagenous (cartilage-like) layer, and elastic filamentous membrane. The collagenous part of the notochord has two main fiber orientations: circular (perpendicular to the main body axis) and longitudinal (parallel to the main axis of the body). Longitudinally organized fibers are located at the outer layer and are the most prevalent. The notochord cylindrical structure gives the body flexibility and support; it also serves as an attachment anchor for segmental muscles and plays a role in cellular-signaling and endodermal structure development.
Cartilage is the main collagen type II containing tissue in the body. There are three major types: 1) elastic cartilage, 2) fibrocartilage, and 3) hyaline cartilage. Elastic cartilage is found in the epiglottis and the eustachian tube. Fibrocartilage often exists temporarily at fracture sites and permanently in the intervertebral disks of the spine, at the mandibular condyle covering in the temporomandibular joint, and in the meniscus of the knee. The hyaline cartilage, also known as articular cartilage, is mostly found in diarthroidal joints covering long bones and it also forms the growth plate for long bones.
Resilient articular cartilage distributes mechanical load and protects the bones from stress. Cartilage dry weight is predominantly collagen type II (stretch resistance) and proteoglycans/GAGs (conveying compression resistance and/or stabilization of fibril-bundle structures). There are also other collagens (types III, IX and XI) and other ECM molecules in cartilage, but in relatively small amounts. Water molecules, organized by proteoglycans in the cartilage meshwork, occupy about 70% of total tissue weight. The main ECM molecules of articular cartilage after collagen type II are dermantan sulfate proteoglycans (DSPGs) (e.g., decorin, biglycan), chondroitin sulfate proteoglycans (CSPG) (e.g., decorin), keratin sulfate proteoglycans (KSPGs) (e.g., aggrecan) and cartilage oligomeric matrix protein, which are involved in collagen network formation and also give the tissue compression resistance, accumulating and holding large amounts of water. The interactions between these compounds and collagen type II are of great interest, due to their relevance to diseases such as osteoarthritis and rheumatoid arthritis.
Rheumatoid arthritis (RA) is a severe disease. The immune system attacks the ECM of joints and causes degradation of articular cartilage. Elevated levels of several antibodies against cartilage ECM components have been detected in serum and synovial fluid of RA patients. However the exact role of these antibodies in initiation and development of the drastic changes in cartilage remains unclear as does the mechanism of tissue destruction. Elevated levels of biglycan antibodies have been detected in the fluids of arthritis patients. Polgar, A. et al., “Elevated levels of synovial fluid antibodies reactive with the small proteoglycans biglycan and decorin in patients with rheumatoid arthritis or other joint diseases.” Rheumatology 42 (2003): 522-527. They are considered to be earlier markers of this disease, and in addition, the presence of collagen type II fibers with irregular diameter and high concentration of collagen cleavage products are also connected to arthritis events. Mitchell, P. G. et al., “Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage,” J. Clin. Biol. 97.3 (1996): 761-768; and Ameye L. et al., “Abnormal collagen fibrils in tendons of biglycan/fibromodulin-deficient mice lead to gait impairment, ectopic ossification, and osteoarthritis,” FASEB J 16(2002):673-680.
There is an ongoing need for additional information on the mechanisms of RA and its effect on collagen in joint tissues. There is a need for a RA model for further study and there is a need for means to rebuild or repair collagen-based ECM in RA patients.